The long-term goal of this project is to clarify the role of antizyme (AZ) in feedback regulation of cellular polyamine transport. It has been well established that cancer cell growth and metastatic potential are closely associated with abnormal polyamine accumulation. The enhanced activity of polyamine transport into cancer cells provides an ideal system for targeting cytotoxic drugs specifically to tumors. Accordingly, many polyamine-based anticancer compounds are being tested and some have progressed to clinical trials. Recent studies have shown that most of these polyamine analogs also stimulate the production of AZ, a regulatory protein that prevents excessive accumulation of polyamines by inhibiting the polyamine transporter. This AZ-mediated limitation of the uptake of potentially useful drugs obviously presents a serious potential problem to the pharmacological use of this transport system. Unfortunately, the mechanisms and even the most basic characteristics of this feedback system are not known. The four investigations in this project are designed to explore fundamental aspects of AZ's involvement in blocking this transporter. The investigations will benefit from the recent discovery that a short N-terminal sequence, present only on the minor, full-length AZ isoform, is critical for maximal transport inhibition. Specifically these studies will (a) establish the relative effects of the two AZ isoforms on both inhibiting polyamine uptake and stimulating export; (b) determine the effectiveness of antizyme inhibitor (AZI) in reversing feedback inhibition of polyamine transport by each of the AZ isoforms; (c) explore possible differences in the interactions of the AZ isoforms with the potential transport intermediate sorting nexin-5; and (d) identify the N-terminal sequences that are important for this feedback activity. The transport inhibition studies will be conducted using CHO clones that have been stably transfected for controlled expression of the distinct AZ isoforms, AZ inhibitor and sorting nexin-5. By elucidating AZ's involvement in controlling this uptake system, this investigation will improve efforts to design and utilize compounds exploiting this useful transporter.